1. Field of the Invention
The present invention relates to a novel member of the serine protease inhibitor (serpin) superfamily of proteins, in particular the plasminogen activator inhibitor (PAI) protein family. More specifically, isolated nucleic acid molecules are provided encoding the pancreas-derived plasminogen activator inhibitor (PAPAI) protein. Plasminogen activator inhibitor polypeptides are also provided. The present invention further relates to methods for treating physiologic and pathologic disease conditions and diagnostic methods for detecting pathologic disorders.
2. Related Art
The mammalian serine protease inhibitors (serpins) are a superfamily of single chain proteins that contain a conserved structure of approximately 370 amino acids and generally range between 40 and 60 kDa in molecular mass. xcex11-Antitrypsin (also known as xcex11-proteinase inhibitor) is a characteristic member of the serpin family in that it is a single chain glycoprotein of nearly 400 amino acid residues that functions by forming a tight 1:1 complex with its cognate protease, neutrophil (leucocyte) elastase, which subsequently slowly dissociates to yield active enzyme and inactive cleaved inhibitor (Carrell, R. W. et al., Cold Spring Harbor Symposia on Quantitative Biology 52:527-535 (1987)). The reactive center of the serpins is typically formed by an X-Ser that acts as a substrate for the target serine protease: xcex11-antitrypsin has a Met-Ser reactive center with the methionine residue providing a putative cleavage site for neutrophil elastase.
The majority of serpins function as protease inhibitors and so are involved in regulation of several proteinase-activated physiological processes, such as blood coagulation, fibrinolysis, complement activation, extracellular matrix turnover, cell migration and prohormone activation (Potempa, J. et al., J. Biol. Chem. 269:15957-19560 (1994)). As noted, serpins inhibit proteolytic events by forming a 1:1 stoichiometric complex with the active site of their cognate proteinases, which is resistant to denaturants (Cohen, A. B. et al., Biochemistry 17:392-400 (1987). The serpins include, but are not limited to, xcex11-antitrypsin (xcex11-proteinase inhibitor), antithrombin III, plasminogen activator inhibitor 1 (PAI-1), plasminogen activator inhibitor 2 (PAI-2), xcex11-antichymotrypsin, and xcex12-antiplasmin (Huber, R. and Carrell, R. W., Biochemistry 28:8951-8966 (1989).
The plasminogen activator system is responsible for the degradation of intravascular blood clots, and also contributes to extra cellular proteolysis in a wide variety of physiological processes of normal development and pathological processes in the etiology of diseases such as tumor invasion and metastasis (Andreasen, P.-A., et al., Int. J. Cancer 72(1):1-22 (1997); Schmitt, M., et al., Thromb. Haemost. 78(1):285-296 (1997)). Plasmin, a trypsin-like protease, is generated from its precursor plasminogen by the action of plasminogen activators, of which there are two types: tissue-type plasminogen activator (also known as tissue plasminogen activator) and urokinase. Plasmin degrades fibrin and several extracellular matrix and adhesion proteins and activates procollagenases.
Plasminogen activation is a highly regulated process. Precise, coordinated, spatial and temporal regulation is afforded by the interaction of a variety of mechanisms. These mechanisms include (1) inhibition by specific plasmin and plasminogen activator inhibitors; (2) binding of plasminogen, plasminogen activators, and inhibitors to fibrin, extracellular matrix proteins, and specific cell surface receptors; (3) release of tissue plasminogen activator and inhibitors from intracellular storage granules; (4) regulation of gene expression of plasminogen activators and inhibitors; (5) an autocrine feedback loop whereby plasmin-mediated activation of latent forms of growth factors regulates the expression of activators and inhibitors; and (6) clearance of free and inhibitor-bound activators via receptors (Bachmann, F. et al., Fibrinolysis, in: Thrombosis Haemostasis 1987, Verstraete, M. et al., eds., Leuven University Press (1987); Danxc3x8, K. et al., Adv. Cancer Res. 44:139 (1985); Pxc3x6llxc3xa4nen, J. et al., Adv. Cancer Res. 57:273 (1991); Vassalli, J. D. et al., J. Clin. Invest. 88:1067 (1991); Carmeliet, P. et al., Thromb. Haemost. 74:429 (1995); Andreasen, P. A. et al., Mol. Cell. Endocrinol. 68:1 (1990); Loskutoff, D. J., Fibrinolysis 5:197 (1991); Keski-Oja, J. et al., Semin. Thromb. Hemost. 17:231 (1991); Blasi, F., BioEssays 15:105 (1993); Andreasen, P. A. et al., FEBS Lett. 338:239 (1994); Bu, G. et al., Blood 83:3427 (1994); and Camani, C. et al., Int. J. Hematol. 60:97 (1994)).
Strong clinical and experimental evidences have suggested a causal role for the tumor-associated urokinase-type PA (u-PA) and the receptor u-PAR in cancer invasion and metastasis (Andreasen, P.-A., et al., Int. J. Cancer 72(1):1-22 (1997); Schmitt, M., et al., Thromb. Haemost. 78(1):285-296 (1997); Duggan, C., et al., Br. J. Cancer 76(5):622-627 (1997)). Consistent with its role in cancer metastasis, overexpression and unrestrained activity of u-PA has been shown to be a prognostic marker in many different types of human cancer (Schmitt, M., et al., Fibrinolysis 6(Suppl. 4):3-26 (1992); Schmitt, M., et al., J. Obstet. Gynaecol. 21:151-165 (1995); Brunner, N., et al., Cancer Treat. Res. 71:299-309 (1994); Kuhn, W., et al., Gynecol. Oncol. 55:401-409 (1994); Ganesh, S., et al., Cancer Res. 54:4065-4071 (1994); Nekarda, H., et al., Cancer Res. 54:2900-2907 (1994); Duffy, M.-J., J. Clin. Cancer Res. 2:613-618 (1996)). The down-regulation of u-PA may occur at the levels of transcriptional regulation of the genes and through interaction with specific endogenous inhibitors such as plasminogen activator inhibitor (PAI).
Only two plasminogen activator inhibitors are known. These are plasminogen activator inhibitor 1 and 2 (PAI-1 and PAI-2, respectively). PAI-1 and PAI-2 regulate mitogenesis, adhesion of myeloid cells, fusion of myoblasts, and migration of endothelial cells (Fazioli, F. et al., Trends Pharm. Sci. 15:25-29 (1995)). Indeed, PAI-1 and PAI-2 are involved in many physiological and pathological processes, including normal pregnancy, preeclampsia, intrauterine growth retardation, wound healing, tumor cell invasion and metastasis, inflammation and arthritis, inflammatory bowel disease, appendicitis, complications from systemic lupus erythematosus, ovulation and prostatic involution and osteonecrosis (Kruithof, E. K. O. et al., Blood 86:4007 (1995)).
Both PAI-1 and PAI-2 have been shown to inhibit extracellular matrix degradation in vitro (Cajot, J.-F., et al., Proc. Natl. Acad. Sci. USA 87:6939-6943 (1990); Baker, M.-S., et al., Cancer Res. 50:4676-4684 (1990)). These results suggest that the inhibitory activity of PAIs might be important in inhibiting tumor malignant progression leading to metastasis. In fact, administration of a recombinant PAI-2 to mice decreases tumor growth (Astedt, B., et al., Fibrinol. 9:175-177 (1995)), whereas overexpression of either PAI-1 or PAI-2 inhibits tumor metastasis (Muller, B., et al., Proc. Natl. Acad Sci. USA 92:205-209 (1995); Soff, G.-A., et al., J. Clin. Invest. 96:2593-2600 (1995)).
In breast cancer, it has been reported that uPA and PAI-i are statistically independent, strong prognostic factors for disease free and overall survival, i.e., high tumor levels are associated with a poor prognosis and are conductive to tumor cell spread and metastasis (Brunner, N., et al., Cancer Treat. Res. 71:299-309 (1994); Duffy, M., et al., Cancer 62:531-533 (1988); Duggan, C., et al., Int. J. Cancer 61:597-600 (1995); Schmitt, M., et al., Br. J. Cancer 76(3):306-311 (1997)). Immunohistochemical staining has detected PAI-I expression at stromal fibroblasts surrounding tumor nodules or at tumor margins (Bianchi, E., et al., Int. J. Cancer 60:597-603 (1995)). The production of PAI-I by the tumor stroma may represent a host defensive response to the excessive proteolysis. In contrast to PAI-I, high level PAI-2 expression may be a favorable prognostic marker in breast cancer (Schmitt, M., et al., Thromb. Haemost. 78(1):285-296 (1997); Duggan, C., et al., Br. J. Cancer 76(5):622-627 (1997)). In breast carcinomas with high uPA values, PAI-2 was associated with a prolonged relapse-free survival, metastasis-free survival, and overall survival (Bouchet, C., et al., Br. J. Cancer 69:398-405 (1994)). In relation to the clinicopathological findings, an inverse correlation between PAI-2 mRNA expression and lymph node metastasis was reported in breast cancers (Sumiyoshi, K., et al., S. Int. J. Cancer 50:345-348 (1992)). In this study, the expression of uPA and PAI-1 was significantly correlated with negative expression of PAI-2; and a low level of PAI-2 expression was significantly associated with lymph node involvement (Sumiyoshi, K., et al., Int. J. Cancer 50:345-348 (1992)). PAI-2 expression is detected predominantly in malignant mammary epithelial cells of primary carcinomas but is also present in stromal cells (Andreasen, P.-A., et al., Int. J. Cancer 72(1):1-22 (1997)). These results indicate that PAI-2 may play a critical role in inhibition of extracellular matrix degradation mediated by plasminogen activator during tumor cell invasion and metastasis.
In view of the wide range of roles that plasminogen activator inhibitors play in physiologic and pathologic processes, there is a continuing need for the isolation and characterization of novel plasminogen activator inhibitors.
The present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding the pancreas-derived plasminogen activator inhibitor (PAPAI) polypeptide having the amino acid sequence is shown in FIGS. 1A-1B (SEQ ID NO:2), FIGS. 4A-4B (SEQ ID NO:13) or the amino acid sequence encoded by the cDNA clone deposited in a bacterial host as ATCC Deposit Number 97657 on Jul. 12, 1996. The nucleotide sequence determined by sequencing the deposited PAPAI clone, which is shown in FIGS. 4A-4B, contains an open reading frame encoding a polypeptide of 405 amino acid residues, including an initiation codon at positions 67-69, with a leader sequence of about 18 amino acid residues, and a deduced molecular weight of about 46 kDa. The amino acid sequence of the mature PAPAI protein is shown in SEQ ID NO:13 (amino acid residues from about 1 to about 387 in SEQ ID NO:13). Another sequence of a PAPAI clone which is shown in FIGS. 1A-1B (SEQ ID NO:1), contains an open reading frame encoding a polypeptide of 392 amino acid residues, including an initiation codon at positions 67-69, with a leader sequence of about 14 amino acid residues, and a deduced molecular weight of about 44.5 kDa. The amino acid sequence of this mature PAPAI protein is shown in SEQ ID NO:2 (amino acid residues from about 1 to about 378 in SEQ ID NO:2).
Thus, one aspect of the invention provides an isolated nucleic acid molecule comprising a polynucleotide having a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence encoding the PAPAI polypeptide having the complete amino acid sequence in SEQ ID NO:2; (b) a nucleotide sequence encoding the PAPAI polypeptide having the complete amino acid sequence in SEQ ID NO:2 but minus the N-terminal methionine residue; (c) a nucleotide sequence encoding the mature PAPAI polypeptide having the amino acid sequence at positions 1-378 in SEQ ID NO:2; (d) a nucleotide sequence encoding the PAPAI polypeptide having the complete amino acid sequence in SEQ ID NO:13; (e) a nucleotide sequence encoding the PAPAI polypeptide having the complete amino acid sequence in SEQ ID NO:13, but minus the N-terminal methionine residue; (f) a nucleotide sequence encoding the mature PAPAI polypeptide having the amino acid sequence at positions 1-387 in SEQ ID NO:13; (g) a nucleotide sequence encoding the PAPAI polypeptide having the complete amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 97657; (h) a nucleotide sequence encoding the mature PAPAI polypeptide having the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 97657; and (i) a nucleotide sequence complementary to any of the nucleotide sequences in (a), (b), (c), (d), (e), (f), (g), or (h) above.
Further embodiments of the invention include isolated nucleic acid molecules that comprise a polynucleotide having a nucleotide sequence at least 95% identical, and more preferably at least 96%, 97%, 98% or 99% identical, to any of the nucleotide sequences in (a), (b), (c), (d), (e), (f), (g), (h), or (i) above, or a polynucleotide which hybridizes under stringent hybridization conditions to a polynucleotide in (a), (b), (c), (d), (e), (f), (g), (h), or (i) above. This polynucleotide which hybridizes does not hybridize under stringent hybridization conditions to a polynucleotide having a nucleotide sequence consisting of only A residues or of only T residues. An additional nucleic acid embodiment of the invention relates to an isolated nucleic acid molecule comprising a polynucleotide which encodes the amino acid sequence of an epitope-bearing portion of a PAPAI polypeptide having an amino acid sequence in (a), (b), (c), (d), (e), (f), (g), (h), or (i) above.
The present invention also relates to recombinant vectors, which include the isolated nucleic acid molecules of the present invention, and to host cells containing the recombinant vectors, as well as to methods of making such vectors and host cells and for using them for production of PAPAI polypeptides or peptides by recombinant techniques.
The invention further provides an isolated PAPAI polypeptide having amino acid sequence selected from the group consisting of: (a) the amino acid sequence of the PAPAI polypeptide having the complete 392 amino acid sequence, including the leader sequence shown in SEQ ID NO:2; (b) the amino acid sequence of the PAPAI polypeptide having the complete 392 amino acid sequence, including the leader sequence shown in SEQ ID NO:2, but minus the N-terminal methionine residue; (c) the amino acid sequence of the mature PAPAI polypeptide (without the leader) having the amino acid sequence at positions 1-378 in SEQ ID NO:2; (d) the amino acid sequence of the PAPAI polypeptide having the complete 405 amino acid sequence, including the leader sequence shown in SEQ ID NO:13; (e) the amino acid sequence of the PAPAI polypeptide having the complete 405 amino acid sequence, including the leader sequence shown in SEQ ID NO:13, but minus the N-terminal methionine residue; (f) the amino acid sequence of the mature PAPAI polypeptide (without the leaser) having the amino acid sequence at positions 1-387 in SEQ ID NO:13; (g) the amino acid sequence of the PAPAI polypeptide having the complete amino acid sequence, including the leader, encoded by the cDNA clone contained in ATCC Deposit No. 97657; and (h) the amino acid sequence of the mature PAPAI polypeptide having the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 97657. The polypeptides of the present invention also include polypeptides having an amino acid sequence at least 95% identical, more preferably at least 96%, 97%, 98% or 99% identical to those above.
An additional embodiment of this aspect of the invention relates to a peptide or polypeptide which has the amino acid sequence of an epitope-bearing portion of a PAPAI polypeptide having an amino acid sequence described in (a), (b), (c), (d), (e), (f), (g), or (h) above. Peptides or polypeptides having the amino acid sequence of an epitope-bearing portion of a PAPAI polypeptide of the invention include portions of such polypeptides with at least six or seven, preferably at least nine, and more preferably at least about 30 amino acids to about 50 amino acids, although epitope-bearing polypeptides of any length up to and including the entire amino acid sequence of a polypeptide of the invention described above also are included in the invention. In another embodiment, the invention provides an isolated antibody that binds specifically to a PAPAI polypeptide having an amino acid sequence described in (a), (b), (c), (d), (e), (f), (g), or (h) above.
For a number of pathologic disorders, such as tumor invasion and metastasis, significant alterations (increases or decreases) in level of PAPAI gene expression can be detected in a sample of tissue or bodily fluid. Increased or decreased levels of PAPAI gene expression can be measured, in such a sample, relative to a xe2x80x9cstandardxe2x80x9d PAPAI gene expression level, i.e., the PAPAI expression level in a tissue or bodily fluid from an individual not having the disorder. Thus, the present invention provides a diagnostic method useful during diagnosis of such disorders, which involves assaying the expression level of the gene encoding the PAPAI protein in tissue or bodily fluid from an individual and comparing the gene expression level with a standard PAPAI gene expression level, whereby an increase or decrease in the gene expression level over the standard is indicative of a pathologic disorder, such as tumor invasion and metastasis, hemorrhage in liver disease, and preeclampsia.
The PAPAI protein inhibits the plasminogen activator system when administered to an individual. The plasminogen activator system is responsible for the degradation of intravascular blood clots, while also contributing to extracellular proteolysis in a wide variety of physiological processes (e.g. wound healing, cell migration, tissue remodeling, angiogenesis, trophoblast implantation, ovulation and fetal development) and pathological processes (e.g. tumor invasion and metastasis, intrauterine growth retardation, preeclampsia, and acute and chronic inflammation). Thus, by the invention, methods are provided for inhibiting the plasminogen activator system, which involve administering an inhibitory amount of PAPAI either alone or together with one or more plasminogen activator inhibitors, such as PAI-I and PAI-2.